Health care product containing isoflavone aglycones and method of producing the same

Abstract

This invention relates to a novel soy isoflavone product with high purities and strong biological activities and the method of producing the same from natural soybeans, soybean materials (i.e. tofu dregs, soy molasses) and other plant sources. The method includes three steps consisting of extraction with an organic solvent, hydrolysis using an acid and crystallization using an antisolvent. The procedure is very simple and thus can be easily adapted for large-scale manufacturing. Moreover, the procedure is able to produce a high yield of total isoflavones at a lower cost. HPLC analysis and E-Screen bioassay reveal that the obtained product not only contains a high content of isoflavone aglycones by weight of dry matter but also exhibits strong estrogenic activity toward human cells. Therefore, the product should be efficacious for relieving menopausal symptoms and other estrogen-deficient diseases and can be used in health care supplements or as additives for foods, beverages or cosmetics.

1. A method of extracting and purifying isoflavone aglycones comprising the steps of:
 a. choosing starting materials that comprise isoflavones; 
 b. grinding said starting materials in step (a) into small particles to obtain a flour; 
 c. extracting said flour obtained in step (b) with an organic solvent having a concentration of 95% to 99.99% to produce an organic solvent extract of isoflavones; 
 d. hydrolyzing said organic solvent extract obtained in step (c) with acid to convert isoflavone glucosides into isoflavone aglycones to produce a hydrolysate; and 
 e. adding water into said hydrolysate obtained in step (d) to increase the volume thereof to form an aqueous mixture and crystallizing said isoflavone aglycones from said aqueous mixture. 
 
     
     
       2. The method according to  claim 1 , wherein said starting materials in step (a) are selected from the group consisting of natural soybeans, soybean materials, lentil, chickpea, split pea, broad bean, kudzu, alfalfa, red clover, subterranean clover, and mixtures thereof. 
     
     
       3. The method according to  claim 1 , wherein said organic solvent for the extraction in step (c) is a solvent selected from the group consisting of ethanol, methanol, and acetone. 
     
     
       4. The method according to  claim 3 , wherein said organic solvent is ethanol. 
     
     
       5. The method according to  claim 1 , wherein said method further comprises the steps of:
 f. mixing said flour obtained in step (b) with said organic solvent to obtain a mixture; 
 g. heating said mixture obtained in step (f); and 
 h. filtering said heated mixture in step (g) to remove undissolved particles to obtain said organic solvent extract of isoflavones. 
 
     
     
       6. The method according to  claim 5  wherein said mixing of solvent and flour in step (f) is conducted at a solvent:flour ratio of 1:1 to 10:1 (mL:g). 
     
     
       7. The method according to  claim 5  wherein said heating in step (g) is conducted at a temperature in the range of 40-90° C. for at least 2 hours. 
     
     
       8. The method according to  claim 5  wherein said method further comprises the steps of:
 i. mixing said organic solvent extract obtained in step (c) with acid to adjust the pH of the resulting mixture to 1-5; and 
 j. heating said mixture obtained in step (i) at a temperature in the range of 40-90° C. for at least 1 hour and obtaining said hydrolysate. 
 
     
     
       9. The method according to  claim 1  wherein said acid for the hydrolysis in step (d) is hydrochloric acid or acetic acid. 
     
     
       10. The method according to  claim 9 , wherein said hydrochloric acid is 37%. 
     
     
       11. The method according to  claim 8  wherein said method further comprises the steps of:
 k. mixing said hydrolysate obtained in step (d) with water to obtain said aqueous mixture; 
 l. cooling said aqueous mixture obtained in step (k) to room temperature or below for at least 10 minutes and precipitating said isoflavone aglycone crystals therefrom; 
 m. filtering said cooled aqueous mixture obtained in step (l) to obtain said isoflavone aglycone crystals; and 
 n. collecting and drying said isoflavone aglycone crystals obtained in step (m) to form an isoflavone aglycone product. 
 
     
     
       12. The method according to  claim 11 , wherein said mixing of hydrolysate in step (k) is conducted at a ratio of 1:1 to 15:1 (mL:mL). 
     
     
       13. The method according to  claim 11  further comprising the steps of:
 o. washing said isoflavone aglycone crystals obtained in step (e) with a solvent; 
 p. re-dissolving said washed product obtained in step (o) in a solvent; 
 q. re-crystallizing said re-dissolved product obtained in step (p) by adding water into said re-dissolved product to form second crystals; and 
 collecting and drying said second crystals obtained in step (q) to form a purified isoflavone aglycone product. 
 
     
     
       14. The method according to  claim 13 , wherein said solvent for washing in step (o) is water; wherein said solvent for re-dissolving in step (p) is 95- 99.99% ethanol. 
     
     
       15. The method according to  claim 1  wherein said hydrolysis step (d) is conducted after said extraction step (c). 
     
     
       16. The method according to  claim 1  wherein the volume of said aqueous mixture increases by 4 to 15 times upon addition of water into said hydrolysate in the crystallization step (e). 
     
     
       17. The method according to  claim 5 , wherein said filtration step is conducted in the absence of ultrafiltration.